ABSTRACT

Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of ‘chatter’ with ER Ca2+ channels. Our data identify new modalities for TRPML1 action.

INTRODUCTION

The remarkable versatility of Ca2+ as an intracellular messenger is underpinned by the spatiotemporal organisation of Ca2+ signals (Berridge et al., 2003). Targeted delivery of Ca2+ to a particular subcellular locale is one way in which Ca2+ can selectively influence a particular cell outcome (e.g. secretion). Equally, global Ca2+ signals, which are often oscillatory, can also be selective through information encoded in oscillation frequency. Interplay between Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from the extracellular space ensures tight control of Ca2+ levels and their downstream targets (Clapham, 2007). Although we have gained much mechanistic insight into the mobilisation of ER Ca2+ stores and the subsequent entry of Ca2+ across the plasma membrane that ensues (Prakriya and Lewis, 2015), we know relatively little about Ca2+ handling by acidic Ca2+ stores such as lysosomes (Patel and Muallem, 2011).

Lysosomes are best known for their degradative role but they maintain an intraluminal Ca2+ concentration (∼500 µM) similar to the ER (Christensen et al., 2002; Lloyd-Evans et al., 2008). They are likely filled by Ca2+-H+ exchange (Melchionda et al., 2016) and express members of the transient receptor potential mucolipin (TRPML) and two-pore channel (TPC) families to effect Ca2+ release (Grimm et al., 2012; Kiselyov et al., 2012; Patel, 2015; Waller-Evans and Lloyd-Evans, 2015). Three TRPML isoforms are present in humans (García-Añoveros and Wiwatpanit, 2014). TRPML1 (encoded by MCOLN1) is ubiquitously expressed and targets to lysosomes through di-leucine motifs (Vergarajauregui and Puertollano, 2006). It is activated by the endo-lysosomal phosphoinositide phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] (Dong et al., 2010) and is a non-selective cation channel permeable to a number of ions including Ca2+ and Fe2+ (Dong et al., 2008). Importantly, mutation of TRPML1 results in the lysosomal storage disorder mucolipidosis type IV (Bargal et al., 2000). This neurodegenerative disorder is characterised by build-up of various lipid species in lysosomes, suggesting a role for TRPML1 in lipid trafficking. It has long been recognised that localised Ca2+ release from the endo-lysosomal system can influence vesicular fusion and fission events (Pryor et al., 2000). Specifically, Ca2+ release through TRPMLs likely underlies functional roles in vesicular trafficking as well as lysosomal exocytosis (Samie et al., 2013), autophagy (Medina et al., 2015) and regulated secretion (Park et al., 2016). Notably, recently described TRPML agonists have proven beneficial in correcting trafficking defects not only in mucolipidosis type IV (Chen et al., 2014) but also in Niemann–Pick type C disease (Shen et al., 2012) and HIV-associated dementia (Bae et al., 2014). Defective TRPML activity has also been implicated in Alzheimer's disease (Lee et al., 2015). Despite their demonstrable (patho)physiological relevance, TRPML-mediated Ca2+ signals are difficult to resolve due to their localised nature. To overcome this, a number of recent studies have used genetically encoded Ca2+ indicators such as GCaMP-3 or GECO fused directly to TRPML1 to record Ca2+ signals in response to agonist activation (Bae et al., 2014; Cao et al., 2015; Medina et al., 2015; Shen et al., 2012). These signals are suggested to reflect local peri-lysosomal Ca2+ release. But it is not clear how the relatively high-affinity Ca2+ indicators used distinguish between local and global Ca2+ signals.

In addition to supporting local Ca2+ fluxes required for endocytic ‘well-being’ (Hockey et al., 2015), lysosomes can also initiate global Ca2+ signals during signalling. This process has been established in the context of the actions of the messenger nicotinic acid adenine dinucleotide phosphate (NAADP) (Galione, 2015). According to the trigger hypothesis, increases in NAADP levels activate endo-lysosomal Ca2+ channels, generating local Ca2+ signals that sensitise neighbouring Ca2+ release channels on the ER. Activation of the latter (on the more substantial store) results in global Ca2+ release. Indeed, in fibroblasts, direct mobilisation of lysosomal Ca2+ stores through osmotic permeabilisation is sufficient to trigger ER-dependent complex Ca2+ signals (Kilpatrick et al., 2013). Physiologically, it is the TPCs that have emerged as the target channels for NAADP (Brailoiu et al., 2009; Calcraft et al., 2009; Patel, 2015) although a role for TRPMLs has also been advanced (Zhang et al., 2011, but see Yamaguchi et al., 2011). Coupling between TPCs and ER Ca2+ channels likely occurs at membrane contact sites between lysosomes and the ER (Kilpatrick et al., 2013; Patel and Brailoiu, 2012; Penny et al., 2014). Although TRPMLs can evoke global Ca2+ entry, at least when mutated as exemplified by the Va mutation in TRPML3 (encoded by MCOLN3) (Grimm et al., 2007; Kim et al., 2007), whether they support lysosome–ER cross talk is not known.

Here, we take advantage of TRPML agonists ML-SA1 (Shen et al., 2012) and MK6-83 (Chen et al., 2014) to probe the Ca2+ permeability of TRPMLs in an intact cell setting. We show that activation of TRPML1 unexpectedly evokes global Ca2+ signals. Mechanistically, we dissect these signals into release and influx components implicating both ER and extracellular Ca2+, in addition to lysosomal Ca2+, for their genesis. Our data suggest that the action of TRPML1 is not limited to local lysosomal Ca2+ signalling.

RESULTS

TRPML agonists evoke global Ca2+ signals

Fusion proteins comprising TRPML1 and either GCaMP-3 (Shen et al., 2012) or GECO (Cao et al., 2015) have been used to resolve presumed local Ca2+ fluxes from lysosomes upon TRPML1 activation. But as shown in Fig. 1A and Movie 1, Ca2+ responses to ML-SA1 (20 µM) were readily resolvable in a proportion of Hela cells loaded with the fluorescent Ca2+ indicator Fura-2. This organic dye distributes throughout the cytosol and thus records bulk changes in cytosolic Ca2+. ML-SA1 responses were concentration dependent (Fig. 1A). Similar results were obtained using primary cultured human fibroblasts. As in HeLa cells, ML-SA1 evoked strong Ca2+ responses in Fura-2-loaded cells in a concentration-dependent manner (Fig. 1B; Movie 2). The amplitude of the Ca2+ signals and the proportion of responsive cells are quantified in Fig. 1C,D. Fibroblasts were more responsive than HeLa cells, although we noted considerable variability between experiments, particularly for fibroblasts (Fig. 1D). To probe specificity, we examined the effects of the new TRPML antagonists ML-SI1 and ML-SI3 (Samie et al., 2013). ML-SI1 could not be used because it evoked Ca2+ signals (Fig. S1A). ML-SI3, however, inhibited ML-SA1-evoked Ca2+ signals (Fig. 1E,F). Collectively, these data suggest that, contrary to the prevailing view, activation of endogenous TRPMLs is capable of evoking global Ca2+ signals.

Subcellular distribution of TRPML1. (A) Confocal fluorescence images (green) of fixed HeLa cells expressing GFP-tagged LAMP1 (left), TRPML1 (middle) or TRPML1D471K (right). Nuclei were stained using DAPI (blue). (B,C) Confocal fluorescence images of cells expressing GFP–TRPML1 or GFP–TRPML1D471K and either colabelled with Lysotracker® Red (LTR) and imaged live (B) or co-transfected with LAMP1-mRFP and imaged following fixation (C). Overlays of images are shown in the right panels where arrows delineate the regions from which red–green intensity plots were derived. Scale bar: 10 µm. (D,E) Summary data of experiments in C quantifying Pearson's correlation coefficients (D) and the percentage of vesicles showing colocalisation (E). Each point represents an individual cell from two independent transfections.

In control experiments, ML-SA1 evoked Ca2+ signals in cells expressing LAMP1 (Fig. 3A). The responses were similar to those in un-transfected cells (Fig. 1). In cells expressing TRPML1, however, there was a large potentiation of the Ca2+ signal (Fig. 3A) such that essentially all of the cells responded (Fig. 3B,C). The potentiating effect of TRPML1 was specific because it was not observed with TRPML1D471K (Fig. 3A–C). Similar results were obtained in fibroblasts (Fig. S1C,D) although this analysis was more limited due to the difficulty in transfecting these cells. Notably, ML-SA1-evoked Ca2+ signals were inhibited in cells expressing TRPML1D471K relative to cells expressing LAMP1 (Fig. 3A–C). The inhibitory effect of TRPML1D471K is likely due to dominant-negative activity resulting from oligomerisation with endogenous TRPML1 (Venkatachalam et al., 2006).

We also tested the effect of the structurally distinct mucolipin analogue MK6-83. Like ML-SA1, MK6-83 evoked large Ca2+ signals in cells expressing TRPML1 but not TRPML1D471K (Fig. 3D,E). Ca2+ signals evoked by both ML-SA1 and MK6-83 in TRPML1-expressing cells were blocked by ML-SI3 (Fig. 3F–I). These molecular and chemical analyses establish the requirement for TRPML1 in global Ca2+ signalling.

TRPML1 couples lysosomal and ER Ca2+ release

To define the Ca2+ sources underlying TRPML1-dependent Ca2+ signals, we first established the relative contribution of store release and Ca2+ entry to the evoked responses. As shown in Fig. 4A,B, removal of extracellular Ca2+ reduced the peak response upon ML-SA1 stimulation and eliminated the sustained phase. ML-SA1-evoked Ca2+ signals in Ca2+-free medium were TRPML1 dependent, as judged by the lack of ML-SA1-evoked Ca2+ signals in TRPML1D471K-expressing cells and upon treatment with ML-SI3 (Fig. 4C). These data suggest that ML-SA1 evokes both Ca2+ release and Ca2+ influx.

We used the lysosomotropic agent Gly-Phe β-naphthylamide (GPN) to disrupt lysosomes. This dipeptide causes osmotic destabilisation of lysosomes and other cathepsin-C-positive compartments (Jadot et al., 1984). GPN treatment resulted in a rapid loss of Lysotracker® red fluorescence consistent with its action on lysosomes (Fig. S1E,F) and inhibited the responses to ML-SA1 indicating a requirement for lysosomes in ML-SA1-evoked Ca2+ release (Fig. 4D). These data are consistent with the lysosomal localisation of TRPMLs (Fig. 2). However, despite the complete compromise of lysosomes upon GPN treatment, the resulting Ca2+ signals were modest relative to those evoked by ML-SA1 (Fig. 4D). This suggested that ML-SA1 did not exclusively release Ca2+ from the lysosomes.

We considered the possibility that activation of TRPMLs might be followed by Ca2+ release from the ER in a manner similar to activation of TPCs by NAADP (Cancela et al., 1999; Patel et al., 2010). To test this, we examined the effects of ML-SA1 after depleting ER Ca2+ stores with thapsigargin. As shown in Fig. 4D, thapsigargin treatment blocked the responses to ML-SA1 indicating a clear requirement for ER Ca2+ stores in ML-SA1 action. To further probe the role of the ER, we used 2-APB to block inositol 1,4,5-trisphosphate (IP3) receptors. As shown in Fig. 4D, 2-APB (100 µM) inhibited ML-SA1-evoked responses. A summary of these data is provided in Fig. 4E. We conclude that activation of TRPMLs on lysosomes triggers Ca2+ release from the ER.

TRPML1 mediates Ca2+ influx

Finally, we examined the nature of the Ca2+ entry pathway evoked by TRPML1 activation. Because store-operated Ca2+ entry is a ubiquitous pathway underlying Ca2+ influx in non-excitable cells, we tested the effects of the Ca2+ entry inhibitor BTP2. BTP2 (20 µM) completely prevented Ca2+ signals evoked by Ca2+ add-back following depletion of ER Ca2+ stores with thapsigargin (Fig. S2A,B). However, BTP2 did not affect the amplitude of Ca2+ signals evoked by ML-SA1 (Fig. 5A,B) although it did slow the rate of rise (Fig. S2C). These data suggest that store-operated Ca2+ entry does not play a major role in Ca2+ signals evoked by TRPML1 activation.

TRPML1 mediates Ca2+ influx. (A,B) Effect of blocking store-operated Ca2+ influx on agonist-evoked Ca2+ responses. (A) Cytosolic Ca2+ levels of TRPML1-expressing HeLa cells stimulated with 20 µM ML-SA1 in the presence of vehicle (0.1% DMSO) or 20 µM BTP2. (B) Summary data of experiments in A where each data point represents an independent repetition. The total number of cells analysed was 151–173. (C–J) Effect of TRPML1 activation on Fe ion entry. HeLa cells expressing TRPML1 were stimulated with 20 µM ML-SA1 (C) or MK6-83 (E) and then 1 mM FeCl2 in the presence of vehicle (0.1% DMSO) or 10 µM ML-SI3. HeLa cells expressing TRPML1D471K were stimulated with either 20 µM ML-SA1 or MK6-83 (G). HeLa cells expressing TRPML1 were stimulated with 20 µM ML-SA1 and either 1 mM FeCl2 or 1 mM FeCl3 (I). Experiments were performed in nominally Ca2+-free medium. Fluorescence signals (denoted F) were recorded after excitation at the isosbestic wavelength of Fura-2 (360 nm) and normalised to that at the beginning of recording (denoted F0). Summary data (D,F,H,J; for C,E,G and I, respectively) quantifying the fractional loss of fluorescence 144s after addition of Fe2+ or Fe3+ under the various conditions (80–169 cells).

The above data raised the possibility that TRPML1 might mediate Ca2+ entry more directly. To test this, we examined the effects of ML-SA1 on Fe2+ entry. Fe2+ permeates TRPML1 and, like several other metal ions, quenches Fura-2 fluorescence (Dong et al., 2008). Fura-2-loaded HeLa cells were therefore challenged with FeCl2 (1 mM) in nominally Ca2+-free medium after stimulation with ML-SA1. FeCl2 induced a significant quench of Fura-2 fluorescence in cells expressing TRPML1 (Fig. 5C). This effect was blocked by ML-SI3 (Fig. 5C,D) and was not observed in cells expressing TRPML1D471K (Fig. 5G,H). Similar results were obtained upon stimulation with MK6-83 (Fig. 5E–H). Challenge with FeCl3 (1 mM) after stimulation with ML-SA1 was without effect (Fig. 5I,J) attesting to specificity. BTP2 slowed Fe2+ entry (Fig. S2D,E), however, add-back of Fe2+ following depletion of Ca2+ stores with thapsigargin did not result in a significant quench (Fig. S2F,G).

DISCUSSION

In this study, we have shown that activation of TRPMLs by two structurally distinct agonists, in both a human cell line and primary cultures, evokes global Ca2+ signals. These data indicate that TRPMLs do not solely mediate local Ca2+ release events from endo-lysosomal compartments. Indeed, we provide evidence that activation of TRPMLs can evoke both Ca2+ release and Ca2+ entry.

We have dissected global Ca2+ release signals into both lysosomal and ER components. There is much evidence supporting the transmission of Ca2+ signals from endo-lysosomes to the ER but to date such ‘chatter’ has been ascribed exclusively to the actions of NAADP and TPCs (Patel and Brailoiu, 2012). Our study extends such coupling to TRPMLs, raising the possibility that other Ca2+-permeable endo-lysosomal channels such as P2X4 receptors and TRPM2 might also functionally couple to ER Ca2+ release channels (Patel and Cai, 2015).

Inhibition of TRPML1 by plasma membrane PI(4,5)P2 is thought to ensure channel activation only in endo-lysosomal compartments (Zhang et al., 2012). In accordance with this idea, basal (unstimulated) whole-cell currents are not detectable in TRPML1-expressing cells. SF-51 (Grimm et al., 2010), the precursor to ML-SA1, however, has been reported to mediate a TRPML1 plasma membrane current (Zhang et al., 2012) consistent with our Ca2+ and Fe2+ entry measurements. Divalent cation entry might therefore result from targeting of a small fraction of TRPML1 to the plasma membrane. Although blockade of store-operated channels reduced TRPML1-dependent Ca2+ and Fe2+ entry (albeit modestly), the amplitude of the Ca2+ signal upon maximal Ca2+ ER store depletion was substantially smaller than that evoked by agonist activation. Indeed, store-operated Fe2+ entry was not demonstrable under our conditions. These data raise the possibility that BTP2 might directly inhibit TRPML1 similar to its effects on TRP-canonical (TRPC) channels (He et al., 2005).

The multi-compartment actions of TRPML1 offer fresh perspective on interpreting Ca2+ signals recorded using Ca2+ reporters fused to TRPML1 (Bae et al., 2014; Cao et al., 2015; Medina et al., 2015; Shen et al., 2012). We suggest that such signals might additionally reflect Ca2+ release from the ER and Ca2+ entry. Indeed, defects in Ca2+ signals reported using such indicators in scenarios of TRPML1 dysfunction are perplexing given that expression of TRPML1 is able to rescue phenotypic defects such as lysosomal storage (Cao et al., 2015; Shen et al., 2012). Use of inert lysosomal proteins for targeting (McCue et al., 2013) and/or low-affinity Ca2+ indicators, to better insulate against bulk cytosolic Ca2+ changes, might aid in isolating local lysosomal Ca2+ release events. Finally, how TRPMLs are endogenously activated remains to be established, but PI(3,5)P2 is a clear candidate raising the possibility that changes in its levels in response to cues such as growth factors might regulate global Ca2+ dynamics.

Cell labelling

All live imaging experiments were performed in HEPES-buffered saline (HBS) comprising (in mM): 1.25 KH2PO4, 2 CaCl2, 2 MgSO4, 3 KCl, 156 NaCl, 10 glucose, and 10 HEPES (pH 7.4; all from Sigma). For measurement of cytosolic Ca2+ concentration and Fe2+ or Fe3+ entry, cells were incubated with Fura-2 AM (2.5 µM) and 0.005% v/v pluronic acid (from Invitrogen) for 1 h. For measurement of lysosome distribution, cells were incubated with Lysotracker® Red (100 nM) (Invitrogen) for 15 min. After labelling, cells were washed with HBS and mounted in a 1-ml imaging chamber (Biosciences Tools) prior to microscopy. For measurement of colocalisation, cells were co-transfected with GFP–TRPML1 or GFP–TRPML1D471K together with LAMP1–mRFP and fixed with 4% paraformaldehyde prior to confocal microscopy. For visualisation of nuclei, cells were incubated with 1 µg/ml DAPI for 5 min.

Epifluorescence microscopy

Epifluorescence images were captured every 3 s with a cooled coupled device camera (TILL photonics) attached to an Olympus IX71 inverted fluorescence microscope fitted with a 20× objective, and a monochromator light source. Fura-2 was excited at 340, 360 or 380 nm, and Lysotracker® Red at 560 nm. Emitted fluorescence was captured using a 440-nm long-pass filter (Fura-2) and 590-nm filter (Lysotracker® Red). TRPML1-expressing cells were identified by monitoring fluorescence of GFP (excitation 488 nm, emission 505 nm). Cells were stimulated with ML-SA1, GW405833 hydrochloride (Sigma; also known as ML-SI1, Haoxing Xu, Department of Molecular, Cellular, and Developmental Biology, University of Michigan, USA, personal communication), ML-SI3 (a kind gift from Haoxing Xu), MK6-83 synthesised as described previously (Chen et al., 2014), GPN (Santa Cruz Biotechnology), BTP2 (Sigma), thapsigargin (Merck), 2-aminoethoxydiphenyl borate (2-APB, Sigma). Where indicated, Ca2+ was omitted from the HBS (nominally Ca2+-free) or replaced with 1 mM EGTA (Ca2+-free). For Fe2+ or Fe3+ quench experiments, cells were stimulated using freshly prepared 100 mM stock solutions of either FeCl2 or FeCl3 (Sigma). The maximal fluorescence ratio change (Ca2+) or the fractional quench at 360 nm excitation (Fe2+ or Fe3+) were quantified on an individual cell basis and averaged for all cells in a given field of view. Experiments were repeated at least three times and all individual data points plotted. The total number of cells analysed is stated in the legends.

Confocal microscopy

Confocal images were captured using an LSM510 confocal scanner (Zeiss) attached to a Zeiss Axiovert 200M inverted microscope fitted with a 63× Plan Apochromat water-immersion objective. DAPI, GFP and Lysotracker® Red or mRFP were excited at 364 nm, 488 nm and 543 nm, and emitted fluorescence was captured using 385–470 nm, 505–550 nm and 560–615 nm band-pass filters, respectively. Pearson's correlation coefficients were calculated from z-stacks (1-µm intervals, 8–12 slices) using the ImageJ plugin Coloc2. The fraction of colocalised vesicles was calculated from the middle slice or slices using the ImageJ plugin SQUASSH.

Footnotes

B.S.K. and E.Y. performed all of the experiments jointly. C.G. synthesised MK6-83. A.H.S. and S.P. conceived the study. S.P. wrote the paper with input from all authors.

Funding

This work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/N01524X/1 to S.P.] and Parkinson's UK [grant number H-1202 to S.P. and A.H.S.]. Deposited in PMC for immediate release.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Similar articles

Other journals from The Company of Biologists

Our resident columnist, Mole, uses Star Wars to illuminate why graduate students feel their future looks like a storm complete with sideways rain, and how to be good mentor rather than an agent of the Dark Side.

iPSCs may be a new source for obtaining HSCs. In their review, Fabiola A. de Castro and colleagues discuss the challenges involved in reprogramming, and the role of microRNAs in iPSC generation and HSC differentiation.

“…one of the best parts of this job: people from different places working together for a common goal.”

Susana and her research group at the Barts Cancer Institute, Queen Mary University of London, investigate how tumour progression is affected by the presence of extra centrosomes in a cancer cell, and how these cancer cells employ mechanisms to adapt to supernumerary centrosomes. She shares her thoughts on the broader impact of her research and the challenges of starting a new lab.